Conservation genomic analysis reveals ancient introgression and
declining levels of genetic diversity in Madagascar’s hibernating
dwarf lemursARTICLE
Conservation genomic analysis reveals ancient introgression and
declining levels of genetic diversity in Madagascar’s hibernating
dwarf lemurs
Rachel C. Williams1,2 Marina B. Blanco1,2 Jelmer W. Poelstra1
Kelsie E. Hunnicutt3 Aaron A. Comeault4,5
Anne D. Yoder1,2
Received: 25 April 2019 / Revised: 15 July 2019 / Accepted: 16 July
2019 / Published online: 21 August 2019 © The Author(s) 2019. This
article is published with open access
Abstract Madagascar’s biodiversity is notoriously threatened by
deforestation and climate change. Many of these organisms are rare,
cryptic, and severely threatened, making population-level sampling
unrealistic. Such is the case with Madagascar’s dwarf lemurs (genus
Cheirogaleus), the only obligate hibernating primate. We here apply
comparative genomic approaches to generate the first genome-wide
estimates of genetic diversity within dwarf lemurs. We generate a
reference genome for the fat-tailed dwarf lemur, Cheirogaleus
medius, and use this resource to facilitate analyses of
high-coverage (~30×) genome sequences for wild-caught individuals
representing species: C. sp. cf. medius, C. major, C. crossleyi,
and C. sibreei. This study represents the largest contribution to
date of novel genomic resources for Madagascar’s lemurs. We find
concordant phylogenetic relationships among the four lineages of
Cheirogaleus across most of the genome, and yet detect a number of
discordant genomic regions consistent with ancient admixture. We
hypothesized that these regions could have resulted from adaptive
introgression related to hibernation, indeed finding that genes
associated with hibernation are present, though most significantly,
that gene ontology categories relating to transcription are
over-represented. We estimate levels of heterozygosity and find
particularly low levels in an individual sampled from an isolated
population of C. medius that we refer to as C. sp. cf. medius.
Results are consistent with a recent decline in effective
population size, which is evident across species. Our study
highlights the power of comparative genomic analysis for
identifying species and populations of conservation concern, as
well as for illuminating possible mechanisms of adaptive phenotypic
evolution.
Introduction
We are in a race against time to preserve species and habitats, and
an urgent goal is to identify those populations that are most
immediately threatened by shrinking geo- graphic distributions and
long-term decreases in population size. Extinction rate estimates
reveal recent and rapid loss of biodiversity, with mammals showing
the highest extinction rates (Ceballos et al. 2015; Ceballos and
Ehrlich 2018). Anthropogenic activity has been identified as a
primary driver of this global environmental change (Dirzo et al.
2014), and leads to habitat loss, overexploitation, and accelerated
climate change (Haddad et al. 2015). The dis- tributions of
non-human primates overlap extensively with a large and rapidly
growing human population, and the unintentional battle for
resources has resulted in an esti- mated ~60% of species being
threatened with extinction and ~75% of populations in decline
(Estrada et al. 2017). The lemurs of Madagascar comprise a clade
that contains
These authors contributed equally: Marina B. Blanco, Jelmer W.
Poelstra
* Rachel C. Williams
[email protected]
1 Department of Biology, Duke University, Durham, NC 27708,
USA
2 Duke Lemur Center, Duke University, Durham, NC 27705, USA 3
Department of Biological Sciences, University of Denver,
Denver, CO 80208, USA 4 Biology Department, University of North
Carolina, Chapel Hill,
NC 27599, USA 5 School of Natural Sciences, Bangor University,
Bangor LL57
2UW, UK
12 34
56 78
roughly 20% of all primate species and starkly illustrates the
negative impacts of anthropogenic activity on non-human primates
(Burns et al. 2016; Salmona et al. 2017): >90% of lemur species
are currently threatened with extinction based on International
Union for Conservation of Nature (IUCN) Red List assessments (IUCN
2019).
Madagascar’s biota is not only largely endemic but also highly
diverse (Goodman and Bernstead 2007). The unique geological and
evolutionary history of the island, combined with the contemporary
degradation of habitats, has led to an urgent need to understand
both their evolutionary history and the ecological interactions
between lemurs and the ecosystems in which they occur
(Albert-Daviaud et al. 2018). For example, quaternary climatic
variation created periods of glaciation worldwide that resulted in
cooler and more arid climates at lower elevations in Madagascar,
whereas periods of glacial minima resulted in warmer and more humid
climates (Rakotoarinivo et al. 2013). These climatic shifts have
been proposed as a driver of speciation in Madagascar (Wilmé et al.
2006), and may have left signatures of population size changes,
divergence and introgression in the genomes of the endemic biota.
Based on broad climatic trends, one prediction is that low-altitude
species would have experienced changes in the location and size of
suitable habitat, this could have led to changes in Ne
that may have left a detectable signature in the genome. Similarly,
we expect that high-altitude species will have had more stable Ne
through time. Our study aims to investigate these
possibilities.
Here, we focus on Madagascar’s dwarf lemurs (genus Cheirogaleus).
The genus has undergone substantial taxo- nomic revisions in recent
years (Rumpler et al. 1994; Pas- torini et al. 2001; Groeneveld et
al. 2009, 2010, 2011; Lei et al. 2014, 2015; Frasier et al. 2016;
Mclain et al. 2017), though four species complexes have remained
generally well resolved: Cheirogaleus sibreei, C. medius, C. cross-
leyi, and C. major. All dwarf lemurs are small (between 150 and 450
g), nocturnal, obligate hibernating primates. They are distributed
across Madagascar and its heterogeneous habitats, from high plateau
to low elevation, in the dry west of the island, the tropical east
(Goodman and Bernstead 2007), and occur sympatrically in many of
these habitats (Blanco et al. 2009). All populations undergo
seasonal hibernation despite living in drastically different
climatic environments, wet to dry, warm to cold, with some species
hibernating for up to 7 months of the year. The hibernation
phenotype is suspected to be ancestral for the clade, although with
subsequent variation in its manifestation (Faherty et al. 2018).
For example, C. sibreei occupies cold environments at high
elevation and hibernates for up to 7 months per year (Blanco et al.
2018), while the sister species C. major and C. crossleyi typically
occur at lower altitudes and hibernate for shorter periods of
time
(~4 months; Blanco et al. 2018). C. medius displays a similar
hibernation phenotype to that of C. sibreei, hiber- nating for up
to 7 months of the year, but in the highly seasonal, dry deciduous
forests of the west.
Conservation efforts must consider current demographics when
designing management strategies for populations in decline
(Harrison and Larson 2014). Effective population size (Ne) is a
well-established parameter for identifying populations at risk
(Nunziata and Weisrock 2018; Yoder et al. 2018), giving insight
into the strength of drift and the degree of inbreeding (Chen et
al. 2016). Genetic diversity is proportional to Ne in a population
of constant size (Kimura 1968), and estimates of changes in Ne
though time can help us better understand potential selective and
demographic forces that have affected present-day populations
(Prado- martinez et al. 2013; Figueiró et al. 2017; Árnason et al.
2018; Vijay et al. 2018). Failure to accommodate for changes in
genetic diversity over time in populations of conservation concern
can lead to elevated extinction risks (Pauls et al. 2013) given
that levels of quantitative genetic variation necessary for
adaptive evolution become reduced, and deleterious mutations can
accumulate (Rogers and Slatkin 2017). Though Ne is typically
considered at the population level, it is also important to assess
how diversity varies among species. Characterizing genetic
diversity in a phylogenetic context requires an informed
understanding of species boundaries, and the very nature of species
bound- aries carries the expectation that phenotypes and genomic
regions remain differentiated in the face of potential
hybridization and introgression (Harrison and Larson 2014).
By investigating the ancestral demography of living species we can
make inferences about their current genetic health and prospects
for survival (Prado-martinez et al. 2013). The development of
methods such as pairwise and multiple sequentially Markovian
coalescent (MSMC) allows for the use of a single biological sample
to give insight into an individual’s ancestry, and theoretically,
to the changes in conspecific Ne over considerable time periods (Li
and Durbin 2011; Schiffels and Durbin 2014). These techniques have
been used to identify specific timescales of species declines, such
as in the case of the woolly mammoth (Mammuthus primigenius)
wherein genomic meltdown in response to low Ne is thought to have
contributed to its extinction (Palk- opoulou et al. 2015; Rogers
and Slatkin 2017). Under- standing the speed of ancestral
population declines, from the reduction in Ne and the accumulation
of detrimental mutations, to genomic meltdown, is critical for the
con- servation of extant species. Importantly, effective popula-
tion size, genetic diversity, and species boundaries can all be
used in conservation planning and, directly used to cal- culate
IUCN ‘Red List' qualification.
A genomic approach offers increased power and preci- sion to
address the questions outlined above. For example,
Conservation genomic analysis reveals ancient introgression and
declining levels of genetic diversity. . . 237
by looking at patterns of genetic variation found within hominid
genomes, numerous studies have revealed instan- ces of
interbreeding and adaptive introgression that occur- red between
early modern humans and archaic hominids (Hajdinjak et al. 2018;
Slon et al. 2018). Admixture between Neanderthals and modern humans
has, for exam- ple, left detectable signatures in the genome
sequences of modern humans (Harris and Nielsen 2016), which can be
identified in the genome sequences of single individuals (Green et
al. 2010a; Hajdinjak et al. 2018; Slon et al. 2018). A focus on
making use of the entirety of information con- tained within a
single genome has also been beneficial for a number of non-human
study systems where the ability to collect and sequence many
samples is prohibitively difficult (Prado-martinez et al. 2013;
Meyer et al. 2015; Palkopoulou et al. 2015; Abascal et al. 2016;
Figueiró et al. 2017; Rogers and Slatkin 2017; Árnason et al.
2018).
Using whole genome sequences for five individuals across four
species of dwarf lemur, we ask if previously published phylogenetic
relationships, based on mitochon- drial and morphological
comparisons (Groeneveld et al. 2009, 2010; Thiele et al. 2013; Lei
et al. 2014), are con- sistent across the genome. We show that
despite an overall congruence, there are small regions of
introgression, a proportion of which is between C. sibreei and C.
sp. cf. medius, two species with the most similar hibernation pro-
files. Since dwarf lemurs are the only obligate hibernating
primates, and hibernation is thought to be ancestral (Blanco et al.
2018), we tested if genes associated with the hiber- nation
phenotype are present in introgressed regions of the genome. We
find evidence that genes implicated in the control of circadian
rhythm and feeding regulation (such as NPY2R and HCRTR2) have
introgressed between species of dwarf lemur.
Together, this work offers the largest contribution of novel
genomic resources for any study to date of Madagascar’s lemurs.
These data allow us to better understand con- temporary levels of
diversity in isolated populations and the demographic history of
Cheirogaleus. Our approach repre- sents a concerted effort to
utilize genomic data for the pur- poses of formulating hypotheses
of conservation threat and to better understand the evolutionary
history of this unique clade of primates, as well as future
prospects for its survival.
Methods
Genome sequencing and assembly
To allow us to compare inter-clade diversity on a genomic scale, we
first generated a reference genome from a Duke Lemur Centre
Cheirogaleus medius individual, DLC- 3619F, which died of natural
causes. DNA was extracted
from liver tissue, and shotgun and Chicago libraries pre- pared by
Dovetail Genomics. Each library was sequenced on two lanes of
Illumina HiSeq 4000 at Duke Sequencing Core (2 × 150-bp reads;
Supplemental Table 1). A de novo assembly was constructed using a
Dovetail’s Meraculous assembler, and the HiRise software pipeline
was used to scaffold the assembly (Putnam et al. 2016). Adaptors
were removed using TRIMMOMATIC (Bolger et al. 2014), which also
discarded reads <23 bp and q < 20. We assessed the
completeness of the assembly using standard summary statistics
(e.g. number of scaffolds and scaffold N50) and the presence of
orthologous genes sequences (BUSCO pipeline; v3; Waterhouse et al.
2017).
To facilitate tests of phylogenetic relationships across the
genome, estimate demographic history, and measure genetic
diversity, we sampled four wild-caught dwarf lemurs, one each from
the species Cheirogaleus sp. cf. medius, C. major, C. crossleyi,
and C. sibreei. Our aim was to include one sample from each of the
four primary clades found within Cheirogaleus (Groeneveld et al.
2010). Individuals were live trapped (sampling locations shown in
Fig. 1a), ear clips taken, and the animals released. C. sp. cf.
medius was sampled from a population in Tsihomanaomby, a forest in
the north-east of Madagascar outside of the known C. medius range,
and where C. sp. cf. medius, C. major, and C. crossleyi are found
in sympatry. Samples from the C. sp. cf. medius population in
Tsihomanaomby are distinct from C. medius in mtDNA (Fig. 1b) and
morphology (Groeneveld et al. 2009). This lineage warrants further
investigation and is here referred to as C. sp. cf. medius, but was
not sampled in the taxonomic revision of Cheirogaleus by Lei et al.
(2014). The C. major individual was sampled from Mar- ojejy
National Park, a rainforest ~45 km from the C. sp. cf. medius
sampling site, which C. crossleyi and C. sibreei also inhabit,
though the latter is only found at elevations above 1500 m.
Sampling sites for C. sp. cf. medius and C. major are separated by
~45 km. We sampled C. crossleyi from Andasivodihazo, Tsinjoarivo
(where C. crossleyi and C. sibreei occur sympatrically) and C.
sibreei from Anka- divory, Tsinjoarivo. Andasivodihazo and
Ankadivory sampling sites are separated by ~6 km but are part of
the same continuous forest (Fig. 1a).
DNA was extracted using DNeasy (C. sibreei) or MagAttract
(remaining samples) kits. One library was pre- pared per individual
and barcoded libraries were then pooled prior to sequencing.
Sequences were generated using the Illumina HiSeq 4000 machine at
Duke Sequencing Core (2 × 150-bp reads), and read quality was
assessed using FASTQC and then filtered using TRIMMOMATIC. Reads
< 50 bp were discarded after trimming for quality (q < 21,
leading: 20, trailing: 20). Expected genome size for each of the
four species we sampled was estimated using JELLYFISH (Marçais and
Kingsford 2011) and FINDGSE (Sun et al. 2018). Reads
238 R. C. Williams et al.
Cheirogaleus medius Cheirogaleus major Cheirogaleus crossleyi
Cheirogaleus sibreei
0.04
84
97
85
99
4
97
76
100
96
99
82
76
98
88
74
97
100
86
99
91
93
Ccro, 106209, Tsinjoarivo
EU825491, Andrambovato/Ambalavero KM872152, Laki
KM872132, Midongy du Sud KM872135, Beharena Sagnira Midongy
KM872138, Ampasy Midongy
Cmed, 106295, Tsihomanaomby
KM872139, Andrafiamena (Anjakely)
EU825469, Kirindy KM872131, Tsingy de Bemaraha
KM872174, Maharira
A B
C
9
Fig. 1 Geographic distributions and phylogenetic relationships of
Cheirogaleus. a Map of Madagascar showing the sampling locations
and IUCN Red List distributions of the four study species. Green
star shows sampling location of outgroup Microcebus griseorufus. b
Maximum likelihood estimation showing relationships within Cheir-
ogaleus based on 684-bp cytochrome c oxidase subunit II (COII).
Individuals used here in genomic analyses are indicated by
their
species colour. Comparative taxa were taken from the National
Center for Biotechnology Information (NCBI) online database.
Locations for these comparative samples were taken from NCBI or, if
unlisted, their publication. Bootstrap values shown. c Cheirogaleus
illustrations, copyright 2013 Stephen D. Nash/IUCN SSC Primate
Specialist Group; used with permission
Conservation genomic analysis reveals ancient introgression and
declining levels of genetic diversity. . . 239
were aligned to the C. medius reference genome using BWA mem (Li
and Durbin 2009) with default settings. Bam files were produced
using SAMTOOLS (Li et al. 2009) and duplicates were marked using
PICARD (Broad Institute 2018, accessed 2018). Reads were then
realigned around indels using GATK’s ‘RealignerTargetCreator’ and
‘IndelRea- ligner’ tools (Mckenna et al. 2010; Depristo et al.
2011). We then genotyped each individual at SNPs mapping to
scaffolds >100 kb (99.6% of the assembly). Individual .gvcf
files were first generated for each individual using GATK’s
‘HaplotypeCaller’ tool. Joint genotyping was then carried out for
all samples using GATK’s ‘GenotypeGVCFs’ tool. The final set of
variants were filtered based on GATK Best Practices (Van der Auwera
et al. 2013). One Microcebus griseorufus individual (RMR66),
sampled from Beza Mahafaly Reserve, was sequenced as ~400-bp insert
libraries on an Illumina HiSeq 3000 (2 × 150-bp reads) to ~40× and
included in the pipeline described above so that it could be used
as an outgroup.
Phylogenetic relationships
To investigate species boundaries between our samples, we first
confirmed the species assignment of our individuals using mtDNA
barcoding. We used cytochrome c oxidase subunit II (COII)
sequencing and NCBI Genbank to access a subset of COII sequences
from across the Cheirogaleus clade. In addition, we used BLAST+
2.7.1 (Altschul et al. 1990) to extract the COII sequences from our
genome-wide Cheirogaleus data. Sequences were aligned using
MAFFT
v7.017 (Katoh et al. 2002), totalling 54 sequences with 684
nucleotide sites. We estimated a maximum likelihood (ML)
phylogenetic tree for the aligned sequences using Mod- elFinder
(Kalyaanamoorthy et al. 2017), as implemented in IQ-TREE v.1.6.6
(Nguyen et al. 2015), to assess relative model fit. The phylogeny
was rooted on the outgroup Microcebus griseorufus and significance
was assessed using 500,000 ultrafast bootstrap replicates (Hoang et
al. 2018).
To assess variation in relationships among species across the
genome, we used SAGUARO (Zamani et al. 2013). SAGUARO generates
similarity matrices for each region that represents a new
relationship, therefore creating statistical local phylogenies for
each region of the genome. We used 105,461,324 SNPs, averaging
approximately four SNPs per 100 bp. Our final set of variants for
the four ingroup species was run in SAGUARO using default
iterations. We visualised the distribution of relationships across
the genome in R (R Core Team 2019), plotting phylogenetic
relationships as neighbour joining trees. Topologies that do not
agree with the previously accepted taxonomic relationships within
the genus (e.g. Thiele et al. 2013; Lei et al. 2014) are from here
termed ‘discordant’ trees. Since the C. medius reference genome is
not annotated, we identified genes in discordant
regions by using BLAST+ to infer gene function from homologous
genes from the grey mouse lemur (Microcebus murinus) NCBI protein
database (Larsen et al. 2017). M. murinus is the highest quality
genome assembly available within the sister clade to Cheirogaleus,
and is ~27My divergent (Dos Reis et al. 2018).
Test for independence
Because SAGUARO identified a small number of discordant genomic
regions, we next carried out formal tests for admixture between
taxa using Patterson’s D-statistics (Green et al. 2010b; Durand et
al. 2011), as implemented in ANGSD (Korneliussen et al. 2014). We
ran two tests; the first test was based on discordant relationships
identified in the SAGUARO analyses, and looked for admixture
between C. crossleyi, C. major, and C. sp. cf. medius, using C.
sibreei as the outgroup (O), given the relationship (((P1, P2),
P3), O). The second test looked for introgression between C. sp.
cf. medius (P2) and C. sibreei (P3) with outgroup Microcebus
griseorufus, and using either C. crossleyi or C. major as P1
(allowing us to test for introgression between different
combinations of species).
Patterson’s D gives a genome-wide estimate of admix- ture and was
not designed to quantify introgression at specific loci (Martin et
al. 2015). To identify specific regions of admixture, we used fd
(Martin et al. 2015), which provides a point estimate of the
admixture proportion at a locus, allowing for bidirectional
introgression on a site-by- site basis. We ran fd on combinations
that had returned a significant Patterson’s D, first between C. sp.
cf. medius and C. major (C. med/C. maj), and secondly between C.
sp. cf. medius and C. sibreei (C. med/C. sib). We used 40-kb
windows with a 10-kb step size.
We defined candidate regions of introgression as those that fell
into the top 0.05% of the genome-wide distribution of fd. These
‘significant’ regions were extracted and BED-
TOOLS (Quinlan and Hall 2010) was used to merge over- lapping
windows. Windows that overran the length of a scaffold were
corrected, reducing the regions of interest from 2.25 to 2.24Mb.
Scaffolds containing windows with significant fd were filtered for
coverage, identifying sites higher than twice the mean, and lower
than half the mean coverage in order to match MSMC2 analyses. This
mean was averaged across samples on a per site basis. Windows were
masked if more than 30% of the sites within a window did not meet
filtering criteria.
Given that the hibernation phenotype is thought to be ancestral for
the dwarf lemur clade (Blanco et al. 2018), we set out to test the
hypothesis that genes associated with hibernation were present in
introgressed regions. Genes present in introgressed regions were
identified from the M. murinus protein database using tblastn, and
filtered by
240 R. C. Williams et al.
evalue(0.0001), max_hsps(1), and max_target_seqs(20). Results were
parsed by bit score, and then evalue to retain only unique
RefSeqIDs. Genes associated with metabolic switches before and
during hibernation were pulled from the hibernation literature
(Faherty et al. 2016, 2018; Grabek et al. 2017) that we refer to as
hibernation-associated genes. These genes were chosen from the
literature on small hibernating mammals and included all gene
expression work in Cheirogaleus. Ensembl biomaRt (Durinck et al.
2005, 2009) was used to convert between different terms of
reference. The hibernation-associated genes were cross- referenced
with genes identified in windows that contained significant fd. To
identify over-represented biological path- ways of introgressed
genes, we ran gene ontology (GO) enrichment analysis using the
R/bioconductor package goseq v.1.32.0 (Young et al. 2010).
Age of introgression
To assess the timing of introgression, we used the software
HYBRIDCHECK v1.0 (Ward and Van Oosterhout 2016). HYBRIDCHECK
differs from fd by using spatial patterns in sequence similarity
between three sequences, as opposed to taking phylogeny into
account by using a fourth sequence as the outgroup. In addition,
the analysis does not use pre- defined windows. As input for
HYBRIDCHECK, we created full-sequence fasta files for each
individual. To do so, we first ran GATK v3.8
FastaAlternateReferenceMaker for each individual, replacing bases
in the C. medius reference genome that were called as non-reference
alleles for that individual, using the unfiltered VCF file (see
genotyping, above). Next, the following classes of bases were
masked: (1) non-reference bases that did not pass filtering (DP
< 5, DP > 2 × mean-DP, qual > 30, QD < 2, FS > 60,
MQ > 40, MQRankSum <−12.5, ReadPosRankSum <−8, ABHet <
0.2 or > 0.8); (2) sites that were classified as non-callable
using GATK v3.8 CallableLoci (using a minimum DP of 3). HYBRIDCHECK
was run for all scaffolds larger than 100 kb using default
settings, testing all possible triplets for the four Cheirogaleus
species. We only retained blocks that contained at least ten SNPs
and had a p-value <1 × 10−6. Blocks identified between sister
species in any given triplet were removed to reduce the chance of
identifying blocks due to incomplete lineage sorting. Estimated
dates for introgressed blocks were converted using a per-year muta-
tion rate of 0.2 × 10−8, based on a per-generation mutation rate of
0.8 × 10−8 (Yoder et al. 2016). This mutation rate is currently the
most accurate estimate available, calculated for mouse lemurs
(Microcebus murinus) from average esti- mates between humans and
mice. As the phylogenetically closest estimate for dwarf lemurs,
this was used throughout the analyses. We checked for overlap
between regions identified using fd and those identified using
HYBRIDCHECK.
Historical and contemporary effective population size
To estimate how Ne has varied through time, for each species, we
estimated Ne using the software MSMC2 (Schif- fels and Durbin 2014;
Malaspinas et al. 2016). Due to only sequencing one individual per
species, we ran each species as a single population of n= 1 (with a
single individual per species, we could not run MSMC2 to estimate
cross- coalescence rates between populations). For this analysis,
we included scaffolds larger than 10Mb, using a total of 52
scaffolds, comprising ~2 Gb and 89.23% of the genome assembly
(total assembly is 2.2 Gb). SAMTOOLS and a custom script from the
MSMC tools repository were used to generate input files per
scaffold: a VCF and a mask file to indicate regions of sufficient
coverage. In addition, a mappability mask was generated using
SNPABLE REGIONS (Li 2009), pro- viding all regions on which short
sequencing reads could be uniquely mapped. To run MSMC2, we used ‘1
× 2+ 30 × 1+ 1 × 2+ 1 × 3’ to define the time segment patterning.
Coalescent-scaled units were converted to biological units using a
generation time of 4 years and a per-generation mutation rate of
0.8 × 10−8. To estimate uncertainty in estimates of Ne, we
performed 50 bootstrap replicates. We calculated the harmonic mean
of the Ne for each individual, excluding both the first five and
the last five time segments.
To study contemporary patterns of genetic diversity, we ran a
sliding window analysis in 100-kb windows across the genomes. As a
measure of heterozygosity, we identified total heterozygous sites
per window, and sites with missing genotype calls in one or more
species. We then divided the number of heterozygous sites by the
window size minus the number of missing genotype calls. We note
that this mea- sure differs from heterozygosity in the
population-genetic sense (H= 2 pq), but is still a useful summary
of the genetic variation contained within the populations from
which our samples were collected.
Results
Genome assembly and sequencing statistics
The final assembly of the dovetail-generated reference genome for
Cheirogaleus medius, based on ~110 × cover- age, comprised 191
scaffolds >100 kb and had a scaffold N50 of 50.63Mb. The genome
contained 92.7% complete single-copy BUSCO orthologs, 3.4% were
fragmented, and 3.2% were missing. For the four wild-caught
individuals, Illumina sequencing produced 350.4 Gb of raw data,
total- ing 1,511,287,743 paired-end reads and representing ~30 ×
coverage per individual. After quality filtering, 1,444,867,212
(97%) high quality reads were retained
Conservation genomic analysis reveals ancient introgression and
declining levels of genetic diversity. . . 241
(82.1% paired, 12.6% forward, 1.7% reverse) and mapped to the C.
medius reference genome. Genome size was estimated to be 2.4 Gb for
C. sp. cf. medius, 2.6 Gb for both C. major and C. crossleyi, and
2.5 Gb for C. sibreei.
Phylogenetic relationships
ModelFinder within IQ-TREE selected model TIM2+F+G4 for the ML
analyses based on the Bayesian information criterion and
corroborated the overall topology as pre- viously published,
confirming the clade-level assignment of the samples we use for
re-sequencing in this study (Fig. 1b). The C. sp. cf. medius
individual phylogenetically groups with individuals identified as
C. medius using mitochondrial DNA (Fig. 1b), and this specific
sub-clade of C. medius was
not sampled by Lei et al. (2014). Our C. major individual
phylogenetically groups into the ‘major C clade’ of Lei et al.
(2014), and C. crossleyi phylogenetically groups with the
‘crossleyi B clade’ of Lei et al. (2014). C. sibreei remains a
single species.
The mitochondrial sequences for the four individuals of C. sp. cf.
medius, C. major, C. crossleyi, and C. sibreei used in this study
are highly divergent, with species separated in well-defined
clades. By using a phylogenomic approach, we were able to confirm
the relationships identified using mitochondrial data at a
genome-wide scale. To do this, we used SAGUARO to identify the most
common relationships among C. sp. cf. medius, major, crossleyi, and
sibreei (the C. medius reference genome was not included in
subsequent analyses due to admixed ancestry) (Williams et al.,
unpublished data). SAGUARO identified 16 unique relation- ships
across the genomes (Fig. 2). We concatenated results that showed
the same topology, and from these remaining distance matrices,
seven (99.4% of the genome) recapitulate phylogenetic relationships
identified using mtDNA. Thus, the vast majority of genomic regions
support previously published relationships among dwarf lemur
species, which were mostly based on mitchondrial data and
morphological comparisons (Groeneveld et al. 2009, 2010; Thiele et
al. 2013; Lei et al. 2014). Our SAGUARO results indicate that C.
major and C. crossleyi are genomically most similar to each other
(sister species cannot be formally inferred with this method, as
trees are unrooted), and equally distant from C. sp. cf. medius. C.
sibreei shows the greatest divergence within the clade, which is in
agreement with previous phylogenetic studies. These results thus
provide the first genome-wide support for four distinct species of
Cheirogaleus.
Additional topologies identified by SAGUARO (Fig. 2) illustrate
variation in local phylogenetic relationships across the genome.
Therefore, despite the overall support for the species tree, local
variation in phylogenetic signal might be driven by evolutionary
processes such as variation in mutation rate, incomplete lineage
sorting, or introgression. For example, SAGUARO identified seven
discordant topolo- gies of which two, spanning 3Mb of the genome in
total, show a close relationship among C. major and C. sp. cf.
medius. Two trees show a topology in which C. sibreei and C. sp.
cf. medius were more closely related (657 kb of the genome), and
three trees grouped C. crossleyi and C. sp. cf. medius as more
closely related (2.4 Mb of the genome). SAGUARO is not forced to
assign genomic regions to all hypotheses and so two of the 16
relationships were not assigned to regions of the genome. Genes
present in dis- cordant regions were identified using tblastn,
resulting in a total of 30 unique genes (Supplementary Table
3).
2205.3 99.53
2.3 0.1Yes
1.7 0.08
1.4 0.06
0.8 0.04
0.6 0.03
0.2 0.01
0.1 0.006
Combined region
length (mb)
% of genome
Discordant? Phylogeny
Yes
Yes
Yes
Yes
Yes
Yes
No
Fig. 2 The relationships assigned to regions of the genome that
were identified in the SAGUARO analyses. Discordance to previously
published phylogenies indicated. Phylogenies are neighbour joining
trees generated from the distance matrices. Phylogenies with the
same overall relationships have been concatenated. Colours
represent spe- cies: Cheirogaleus sp. cf. medius (blue), C. major
(red), C. crossleyi (purple), and C. sibreei (yellow)
242 R. C. Williams et al.
Signals of ancient introgression
To test if discordant trees were a result of admixture between
species, as opposed to incomplete lineage sorting, we used
Patterson’s D-statistics. When testing for intro- gression between
C. sp. cf. medius with either C. crossleyi or C. major, we found a
genome-wide excess of ABBA sites, giving a significantly positive
Patterson’s D of 0.014 ± 0.0008 (Z score 16.94) between C. sp. cf.
medius and C. major, thus providing evidence for admixture between
C. major and C. sp. cf. medius. This result is also in agreement
with the most common discordant topology identified in the SAGUARO
analysis summarized above. When
testing for introgression between C. sp. cf. medius and C. sibreei,
we again found a genome-wide excess of ABBA sites. With C.
crossleyi as P1, there was a significantly positive Patterson’s D
of 0.06 ± 0.001 (Z score 54.37). With C. major as P1, there was a
significantly positive Patter- son’s D of 0.06 ± 0.01 (Z score
53.89). Both results are evidence for introgression between C. sp.
cf. medius and C. sibreei.
To identify specific regions of admixture, we used the fd statistic
(Martin et al. 2015; Fig. 3). We calculated fd for comparisons that
had significant genome-wide estimates of Patterson’s D: between C.
sp. cf. medius and C. major (C. med/C. maj), and between C. sp. cf.
medius and C. sibreei
Fig. 3 Fd test for introgression plotted across the genome. Fd
statistic computed in 40-kb windows with a 10-kb sliding window,
with the full phylogeny and associated test indicated on the right.
Scaffolds are arranged from largest to smallest, alternating in
colour. ‘Significant regions' are shown in red. Hibernation-related
genes are labelled. a
Results from a test of introgression between Cheirogaleus sp. cf.
medius and C. major (C. med/C. maj). b Results from a test of
introgression between C. sp. cf. medius and C. sibreei (C. med/C.
sib). c Isolated scaffolds of interest
Conservation genomic analysis reveals ancient introgression and
declining levels of genetic diversity. . . 243
(C. med/ C. sib). We define a region as being introgressed if fd
was in the top 0.05% of the genome-wide distribution. The C. med/C.
maj test (Fig. 3a) resulted in an average of 1903 biallelic SNPs
per window, a mean fd of 0.0008 and a 0.05% cutoff of 0.137. There
were 21 scaffolds containing one or more regions of significant fd.
The scaffold with the highest average fd was
ScMzzfj_1392_HRSCAF_67928 (fd= 0.01). The C. med/C. sib test (Fig.
3b) averaged 3005 biallelic SNPs per window, with an average fd of
0.01 and a 0.05% cutoff of 0.188. There were 23 scaffolds with
regions of one or more significant fd window, and scaffold
ScMzzfj_2194_HRSCAF_93304 had the highest average (fd= 0.07).
We next used HYBRIDCHECK to estimate the date of the introgression
within the clade (Fig. 4). The average age of introgressed regions
across all species pairs was 4.12My, and the average length of
introgressed regions was 2.2 kb (Table 1), showing that
introgression was predominantly ancient. The lower boundary
estimate of the most recent split within the Cheirogaleus clade is
~6Mya (identified in Dos Reis et al. (2018) between C. medius and
C. major) and therefore approaches, but does not overlap with, our
oldest
average estimate of admixture, which is between C. major and C.
sibreei and dated 4.52Mya. Following the removal of sister species
in any given triplet, the number of intro- gressed regions were
calculated between pairs from any given triplet. The largest number
of introgressed regions (totaling 9553) were detected between C.
sp. cf. medius and C. sibreei, generated from testing triplet
‘'med/cro/sib'’ and ‘med/maj/sib’. These values were followed by
regions detected between C. sp. cf. medius and C. major (4959).
These results corroborate our fd findings. Calculating the overlap
between fd and HYBRIDCHECK blocks for C. med/C. maj showed that 8
of the 86 fd blocks overlap with the 4959 HYBRIDCHECK blocks to
create a total of 12 discrete over- lapping sections. For C. med/C.
sib, 26 of the 101 fd blocks overlap with the 9553 HYBRIDCHECK
blocks to create a total of 130 discrete overlapping sections. The
larger number of blocks identified by HYBRIDCHECK, compared with fd
stat, is likely due to the fact that HYBRIDCHECK may incorrectly
identify small regions of incomplete lineage sorting as
introgressions.
Genes within introgressed regions
Parsed RefSeq ID BLAST results were filtered by bit score and
evalue, resulting in 4668 hits, containing 660 genes for C. med/C.
maj and 4605 hits, containing 676 genes, for C. med/C. sib.
However, hibernation genes were not more likely to be in
introgressed regions that we would expect by chance for the C.
med/C. maj test, in fact there was a slight deficit of hibernation
genes in introgressed regions, relative to the total number of
hibernation genes (χ2= 5.55, df= 1, p= 0.018). Nonetheless,
introgressed regions for between C. medius and C. major contained
3% of all hibernation genes (21 unique genes; Supplementary Table
3). The C. med/C. sib test also did not contain more hibernation
genes
4
8
12
16
4.3 MY
3.9 MY
Past Present
BAFig. 4 Ancient introgression between C. sp. cf. medius, C. major,
C. crossleyi, and C. sibreei. a Overall relationships within the
clade and the approximate mean timing of introgression. Thicker
dashed line indicates a larger proportion of admixed regions. b The
age of introgressed regions between species pairs
Table 1 Tract sizes and estimated dates of introgression
C. sp. cf. medius
C. sp. cf. medius
C. sibreei 3,945,108 4,519,688 4,135,655 –
The mean size of introgressed regions in base pairs is shown above
the diagonal and the estimated mean age of introgressed regions in
years below the diagonal
244 R. C. Williams et al.
than expected by chance (χ2= 3.11, df= 1, p= 0.078) and
introgressed regions here contained 3.6% of total hiberna- tion
genes (25 unique genes; Supplementary Table 3). Results do not
account for the fact that genes may be non- randomly clustered
because annotations are not yet avail- able for the full
genome.
GO analyses did detect 30 and 32 GO categories that were
significantly over-represented in the introgressed regions (p <
0.00006, p < 0.00009) in the C. med/C. maj and C. med/C. sib
tests, respectively (Supplementary Table 4). Among the
significantly over-represented GO categories were a large
proportion of categories related to transcrip- tional activity,
which is a critical component for inducing the hibernation
phenotype (Morin and Storey 2009). Moreover, several genes that
appear to have introgressed between C. medius and C. sibreei are
more directly relevant to hibernation (Fig. 3c). These include
genes relating to circadian rhythm, the regulation of feeding
(NPY2R; Soscia and Harrington 2005), (HCRTR2; Kilduff and Peyron
2000)), and macrophage lipid homoeostasis (ABCA10; Wenzel et al.
2003).
Historical and contemporary effective population size
To understand how historical demographic events have influenced
effective population size (Ne) through time, we reconstructed the
demographic history of each species based on a multiple
sequentially Markovian coalescent model (Fig. 5a). From our
results, we infer that Cheirogaleus sp. cf. medius has seen two
expansions in Ne in the last 2 million years, followed by a decline
at ~300 kya, with an overall harmonic Ne mean of 92,285
individuals. Cheir- ogaleus major shows an initial peak over two
million years ago, followed by a decline and then a more moderate
Ne
throughout the last 200 ky, averaging overall Ne as 98,005. This
trajectory is very similar to that shown by Cheir- ogaleus sibreei,
which shows the most stable Ne of the Cheirogaleus species tested
and a relatively high harmonic mean of 103,630. Finally, C.
crossleyi shows a relatively large increase in Ne to almost 200 k;
this change occurs at a similar time that we see the decline in C.
sp. cf. medius. Contemporary heterozygosity is consistent with the
MSMC2 results and allows us to infer that the C. sp. cf. medius
population has not recovered from the decline 300 kya (Fig. 5a).
Similarly, the effects of a large increase in Ne seen in C.
crossleyi are still evident today; the harmonic mean estimate is
the highest of the four species, at 108,229 individuals.
We next quantified heterozygosity for each individual in our data
set to test whether long-term population size is correlated with
contemporary levels of genetic diversity. We assessed levels of
heterozygosity in sliding windows across the genome. The C. sp. cf.
medius individual inclu- ded in our study has markedly lower
heterozygosity than the other three species (genome-wide average=
0.001 versus 0.003, 0.003, and 0.004; Fig. 5a), while C. major and
C. sibreei both show immediate levels of current hetero- zygosity
(both average 0.003; Fig. 5b). C. crossleyi appears to have had a
much larger Ne for ~150 ky, and this is reflected in it having the
highest level of genome-wide heterozygosity (0.004).
Discussion
Our study not only confirms relationships between Mada- gascar’s
rarely seen nocturnal hibernators, but also identifies ancient
introgression that has occurred between species. By utilizing a
combination of whole genome sequencing, Markovian coalescent
approaches, and phylogenomic ana- lyses of four species of
Cheirogaleus, we have been able to examine the demographic history
of dwarf lemurs in greater resolution than has yet been attempted.
We confirmed previously hypothesized phylogenetic relationships
among four species within the genus, calculated
heterozygosity
5 10 20 50 100 200 500 1k 2k
0 50
10 0
15 0
20 0
25 0
30 0
A
B
Fig. 5 Estimated population sizes and levels of diversity. a
Effective population size (Ne; y-axis) estimated for each species
over the last 2 million years (x-axis). The bold line represents
the MSMC2 estimate for the original data, and the surrounding lines
in the corresponding species colours represent 50 bootstrap
replicates. Present time is at the origin and time into the past
along the x-axis. b Heterozygosity cal- culated in 100-kb windows
across the genome
Conservation genomic analysis reveals ancient introgression and
declining levels of genetic diversity. . . 245
levels, and assessed historical effective population sizes, thereby
using different measures of diversity to assess conservation needs.
Surprisingly, these analyses also iden- tified regions of
introgression among species that contained several significantly
over-represented GO categories.
Functional implications of ancient admixture for the hibernation
phenotype
We identified regions of admixture between C. sp. cf. medius and
two other species, C. major and C. sibreei. Contemporary or even
relatively recent gene flow between C. medius and C. major would
seem unlikely given the long divergence time (Dos Reis et al.
2018), current morpholo- gical differences (including a threefold
difference in weight), and the disjunct geographic ranges (dry
western forest versus eastern tropical rainforest) of C. medius and
C. major (Goodman and Benstead 2003; IUCN area of occu- pancy
distributions shown in Fig. 1a). However, the C. sp. cf. medius
individual characterized by this study was sam- pled from
Tsihomanaomby, a forest outside of the typical range of the species
where it occurs sympatrically with a population of C. major. As
would be expected given the age of lineage diversification, these
introgressed tracts are much smaller than the more recently admixed
human genome, where introgressed haplotypes average 50 kb
(Sankarara- man et al. 2016; Browning et al. 2018). Both the small
tract lengths (on average 2.2 kb) and estimates of admixture timing
based on sequence divergence indicate that admix- ture between the
species was ancient. Specifically, estimates of the timing of
admixture suggests that admixture occurred not long after
divergence of the species (Dos Reis et al. 2018).
Building on these estimations of ancient admixture, introgression
between Cheirogaleus medius and C. sibreei may have more biological
relevance for understanding their life history. They are the two
‘super hibernators’ in the clade and have similar hibernation
profiles relative to the other two species, consistently showing
the longest duration of torpor (Blanco et al. 2016, 2018). Both
species hibernate for the longest periods of time (up to 7 months
of the year) and in the more extreme environments: C. medius can
hibernate under extreme fluctuations in daily ambient tem-
peratures up to 35 °C, whereas C. sibreei is a high elevation
species that hibernates in the coldest habitats of Mada- gascar,
with an average body temperature of 15 °C (Blanco et al. 2018).
They are not sister species, and are estimated to have diverged
from the most recent common ancestor 18Mya (Dos Reis et al. 2018).
Hibernation within the clade is hypothesized to be ancestral
(Blanco et al. 2018), there- fore, the adaptive introgression of
genes relating to hiber- nation is plausible but would have
required a strong selective advantage for these regions in the
recipient
population. Confirmation of adaptive introgression would require
additional evidence, such as population-level data showing evidence
for a sweep of the introgressed allele (Arnold et al. 2016). The
significant GO categories relating to transcriptional activity are
particularly interesting because of the transcriptional control
mandatory for initi- ating and maintaining the hibernation
phenotype. Entry into torpor requires coordinated controls that
suppress and rep- rioritize all metabolic functions including
transcription (Morin and Storey 2009).
We found several genes associated with circadian rhythms to be
located in introgressed regions. Gene HCRTR2 plays a role in the
regulation of feeding and sleep/ wakefulness (Kilduff and Peyron
2000) and was located in both tests (C. med/C. maj and C. med/C.
sib). NPY2R is a gene that was identified to be upregulated in the
fat versus torpor phases of Faherty et al. (2018); NPY2R was
identified in an introgressed region between C. med/C. sib.
Circadian clocks play an important role in the timing of mechanisms
that regulate hibernation (Hut et al. 2014), as does the cir-
cannual clock with its impact on seasonal timing (Helm et al.
2013). The role of the circadian rhythm through the duration of
hibernation in mammals is unclear, with arctic ground squirrels
(Urocitellus parryii) showing inhibition of circadian clock
function during hibernation (Williams et al. 2017). Neuropeptide Y
has been shown to modulate mammalian circadian rhythms (Soscia and
Harrington 2005) and there has been interest in its potential for
anti- obesity drug development (Yulyaningsih et al. 2011) due to
increased expression exerting anorexigenic effects (Yi et al.
2018). Thus, the upregulation of NPY2R during the process of
hibernation and the highly conserved nature of the Y2 receptor
warrants further investigation for its application to human
medicine. Also of interest, ABCA10 was identified in Faherty et al.
(2016) to be differentially expressed between ‘active1’ and
‘torpor’ states (‘active1’ represents the accu- mulation of weight
during the summer months). This gene is shown to be present in
introgressed regions in the C. med/ C. sib test. ABCA10 plays a
role in macrophage lipid homoeostasis (Wenzel et al. 2003). Body
fat reserves are essential to enter hibernation and are then
metabolised and depleted throughout the season (Blanco et al.
2018). As longer hibernators, C. medius and C. sibreei would be
expected to need greater control over fat metabolism.
Climatic impacts on population size through time and prospects for
the future
Madagascar’s history of climatic variation has shaped the fauna of
the island (Dewar and Richard 2007). Fluctuation of climate such as
lower temperatures at the Eocene–Oligocene transition (Dupont-Nivet
et al. 2007), followed by warming climate and low precipitation of
the
246 R. C. Williams et al.
Miocene, caused dramatic contractions and expansions of vegetation
(Hannah et al. 2008). Climatic variation has been incorporated into
models that try to explain the micro- endemism and historical
patterns of dispersal and vicariance across the island (Wilmé et
al. 2014). Martin (1972) posited a model that considered larger
rivers as geographical barriers to gene flow, suggesting that
climatically and physically defined zones can operate as agents for
geographical isola- tion and speciation, such as climatic pulsing.
Wilmé et al. (2006) followed a similar theme looking at watersheds
in the context of quaternary climatic and vegetation shifts,
result- ing in patterns of diversity and microendemism. Craul et
al. (2007) tested the influence of inter-river-systems on simul-
taneously isolating populations and providing retreat zones.
The changes in Ne seen in Fig. 5a fit with Wilmé’s hypothesis of
climatic pulsing. During periods of glaciation, when the climate
was cooler and drier, high-altitude species were likely buffered
from changing conditions, whereas low-altitude species likely
experienced declines associated with aridification and greater
levels of habitat isolation, and may have expanded during glacial
minima, when climatic conditions were warmer and more humid. The
finding of a more stable Ne through time for C. sibreei, the high
plateau specialist, supports the theory that populations at high
alti- tudes avoided the expansions and contractions occurring at
lower elevations. Results for species typically found at lower
elevations show more variation, e.g. a decrease in C. sp. cf.
medius Ne occurs simultaneously with an increase in C. crossleyi
Ne. The inference of demographic history is strongly influenced by
population structure and changes in connectivity (Hawks 2017; Song
et al. 2017), and so we are careful to discuss overall trends and
not specifics. Results show an overall decline for all species in
the last 50 k years. Declines in more recent times are echoed in
great apes, including modern humans (Prado-martinez et al. 2013).
While extremes at either end of the time scale in MSMC2 should be
taken with caution (Hawks 2017), it appears that certain dwarf
lemur populations have been declining for as long as 300 ky.
Furthermore, these declines are well supported by the bootstrap
values, particularly in the case of C. sp. cf. medius.
Our results indicate that population declines of dwarf lemurs
occurred long before the arrival of humans, intro- ducing the
possibility of long-term low Ne. As these are all species of
conservation concern, we assessed genomic levels of heterozygosity
to derive an estimate of genetic diversity. C. sp. cf. medius had
the lowest overall hetero- zygosity of the samples here examined,
and these values are indicative of a small and isolated population
at risk of inbreeding depression (Rogers and Slatkin 2017). The C.
sp. cf. medius individual examined here was collected from
Tsihomanaomby in the north-east of Madagascar. This is a
sub-humid rainforest site (~7 km2) not typical to the C. medius
habitat preference given that the species is normally associated
with the dry forests of the west (Groves 2000; Hapke et al. 2005;
Groeneveld et al. 2010, 2011). Inter- estingly, our estimates of
historic effective population size trajectory suggest that the Ne
of C. sp. cf. medius has long been much lower than that of other
dwarf lemurs (Fig. 5a). It is therefore possible that the decline
in C. sp. cf. medius Ne seen ~300 kya is the time that this
population became isolated from other C. medius populations, or a
consequence of a bottleneck associated with their colonisation of
Tsi- homanaomby. It is plausible that the Tsihomanaomby population
was founded by individuals from the north west (from which it now
appears separated; Fig. 1b), and that founder effects are still
impacting the present-day popula- tion, indicated the by low
heterozygosity in our C. sp. cf. medius individual (Fig. 5b).
Summary
Our study illustrates the power of selective sampling and genomic
analysis for identifying populations/species in need of
conservation, given that analyses of whole genomes allows us to
identify historical events detectable in the genomes of extant
individuals. We conclude from our analyses that the Cheirogaleus
sp. cf. medius population warrants further investigation, both
taxonomically, given its disjunct distribution, and as a species of
conservation con- cern. All four taxa show temporal changes in
ancestral Ne
over the last 2 million years with an overall decrease in Ne
in recent years (50 k years). While species may recover from some
fluctuation in population size, large crashes leave long-term
traces in the genome that can be detrimental to long-term survival
of populations (Palkopoulou et al. 2015). We demonstrate that for
Cheirogaleus, such events can be potentially disadvantageous, such
as low hetero- zygosity from population declines, as seen in C. sp.
cf. medius. However, the significant enrichment of several
categories of genes in introgressed regions of the genome, shown by
the GO analysis, demonstrates for the first time a potential source
of novel variation in Cheirogaleus. Hybridization is a pervasive
evolutionary force and can drive adaptive differentiation between
populations (Rune- mark et al. 2018). We show that ancient
admixture may have been a possible mechanism for adaptive
phenotypic evolution in these species of concern.
Data archiving
Genomic sequences can be found under NCBI BioProject accession
PRJNA523575. BioSample accessions are listed in Supplementary Table
1.
Conservation genomic analysis reveals ancient introgression and
declining levels of genetic diversity. . . 247
Acknowledgements We thank Stephen D. Nash for the use of his
Cheirogaleus illustrations. We thank Prof. Manfred Grabherr and
Jessica Rick for advice on analyses, and Michelle Larsen for her
time in the lab. We thank Rikki Gumbs and three anonymous reviewers
for comments on the manuscript. We thank the Malagasy authorities
for permission to conduct this research. This study was funded by a
National Science Foundation Grant DEB-1354610 and Duke Uni- versity
startup funds to ADY. RCW and ADY also received a Matching Funds
Award from Dovetail Genomics for the reference genome. ADY
gratefully acknowledges support from the John Simon Guggenheim
Foundation and the Alexander von Humboldt Founda- tion during the
writing phase of this project. We are grateful for the support of
Duke Research Computing and the Duke Data Commons (NIH
1S10OD018164-01). This is Duke Lemur Center publication no.
1437.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict
of interest.
Publisher’s note: Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional
affiliations.
Open Access This article is licensed under a Creative Commons
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Conservation genomic analysis reveals ancient introgression and
declining levels of genetic diversity. . . 251
Conservation genomic analysis reveals ancient introgression and
declining levels of genetic diversity in Madagascar’s hibernating
dwarf lemurs
Abstract
Introduction
Methods
Results
Phylogenetic relationships
Discussion
Functional implications of ancient admixture for the hibernation
phenotype
Climatic impacts on population size through time and prospects for
the future
Summary